Pharmaceutical compositions for competitively inhibiting the binding of a retrovirus to the IFN-receptor and means for diagnosis of an HIV infection

ABSTRACT

The present invention is based on the observation that HIV does not only interact with the CD4 receptor of target cells but that there exists a different type of interaction between HIV envelope protein and the IFN receptor of the target cell. Thus, blocking such interaction can be useful for preventing or treating retroviral infections. Accordingly, the present invention relates to pharmaceutical compositions for competitively inhibiting the binding of a retrovirus, preferably HIV, to the IFN-receptor of a target cell. Preferably, said pharmaceutical compositions comprise a protein, polypeptide or equivalent molecule or a combination thereof which binds to the IFN-α/β-receptor or to HIV-gp41. Furthermore, the present invention relates to the use of said protein, polypeptide or equivalent molecule or IFN-β or a combination thereof for the preparation of a pharmaceutical composition for preventing or treating retroviral infections. The present invention also relates to a combination of substances for the diagnosis of an HIV infection which is based on the detection of the interaction between HIV-gp41 and the IFN-α/β-receptor.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/EP 96/03648 which has an Internationalfiling date of Aug. 19, 1996 which designated the United States ofAmerica, the entire contents of which are hereby incorporated byreference.

The present invention relates to pharmaceutical compositions forcompetitively inhibiting the binding of a retrovirus, preferably HIV, tothe IFN-receptor of a target cell. Preferably, said pharmaceuticalcompositions comprise a protein, polypeptide or equivalent molecule or acombination thereof which binds to the IFN-α/β-receptor or to HIV-gp41.Furthermore, the present invention relates to the use of said protein,polypeptide or equivalent molecule or IFN-β or a combination thereof forthe preparation of a pharmaceutical composition for preventing ortreating retroviral infections. The present invention also relates to acombination of substances for the diagnosis of a HIV infection which isbased on the detection of the interaction between HIV-gp41 and theIFN-α/β-receptor.

Since the discovery of the human immunodeficiency virus (HIV) as theetiological agent of AIDS, there have been many advancements in theunderstanding of the replicative cycle of HIV and in the design ofantiviral drugs. The development and application of such antiviralpharmaceutical compositions that inhibit the replication of HIV arebased on the knowledge of the replicative cycle of HIV: The first stepin HIV replication is access of the retrovirus to the organism throughexposure to HIV-infected blood or body fluids. The envelope glycoproteingp120 which plays a major role, selectively binds to it's receptor CD4,a cell-surface protein located on a subpopulation of helper Tlymphocytes. After binding, the HIV virus enters the susceptible cells.The virus is uncoated within the cytoplasm of these cells yielding viralgenomic RNA which is transcribed by virus-encoded reverse transcriptaseinto single-stranded DNA. Then, this single-stranded DNA is duplicated,and, after degradation of the RNA strand by ribonuclease H, proviral(unintegrated) circular double stranded DNA is formed. The proviral DNAcan then migrate into the nucleus of the host cell and become integratedinto the genome. After a latency period RNA polymerases of the host celltranscribe the DNA of the integrated HIV into mRNA, which is thentranslated into viral proteins. After translation the precursor proteinsare further modified (specific cleavage by virus-specific proteins andglycosylation by host enzymes) and the viral proteins are assembled inthe cytoplasm. After budding of the virus from the host cell surfaceadditional cells are infected and the cycle is repeated.

Known targets in the replicative cycle of HIV for therapeuticintervention include binding of the target cell and entry (previousagents: soluble CD4, SCD4-PE40), reverse transcription of the retrovirus(inhibitors: Zidovudine, Nevirapine etc.), transcription and translation(inhibitors: Trichosanthin, tat-antagonists, anti-sense oligonucleotidesetc.) and viral maturation and budding (inhibitors: N-butyl-DNJ,protease inhibitors).

Furthermore, interferons (IFN) are used for therapeutic intervention ofHIV infection, since they interact on different levels of the HIVreplication cycle (reviewed in Pitha, Antiviral Res. 24 (1994),205-219).

However, although progress has been made in developing drugs andtherapies for treating, retroviral, for example, HIV infections,effective pharmaceutical compositions without severe side effects arestill lacking.

Thus, the technical problem underlying the present invention is toprovide new compounds for the prevention, treatment and diagnosis ofretroviral, preferably HIV, infections which overcome the disadvantagesof the prior art compounds.

The solution to said technical problem is achieved by providing theembodiments characterised in the claims. It has been, surprisingly,found that a retrovirus does not only interact with the CD4 receptor oftarget cells but that there exists a different type of interaction,namely between the IFN-receptor of the target cell and retroviralenvelope protein(s) which seems to be independent of theCD4-interaction. Thus, HIV should be able to enter and infect every celltype, since IFN receptors are located on all cells. Accordingly, thepresent invention relates to a pharmaceutical composition, comprising aprotein, peptide or a functionally equivalent molecule or a combinationthereof which competitively inhibits the binding of a retrovirus to theIFN-receptor of a target cell and optionally a pharmaceuticallyacceptable carrier.

In this context, the term "functionally equivalent molecule" refers toany molecule, which is useful as a competitive inhibitor in order toprevent or at least reduce interactions between the IFN-receptor and theretrovirus. Such "functionally equivalent molecules" include, forexample any other IFN- or HIV-gp41-binding molecule of peptidic ornonpeptidic character, e.g. any molecule developed by conventional drugdesign methods according to the basic principles of bindinginteractions. The term "competitively inhibits", as used herein, relatesto any molecular interaction that competitively inhibits the binding ofa retrovirus to the IFN-receptor of the target cell, for example, bybinding to the receptor, binding to the responsible surface protein ofthe retrovirus, by receptor independent binding to the target cell, bybinding to other sections of the virus surface, by altering the receptoror by altering the responsible surface protein of the retrovirus.

IFN receptors are described in detail in the following publications:Mariano et al. in: Interferon: Principles and Medical Applications,Baron et al. (eds.) UTMB, Galveston, Tex. 1992 pp. 128-138; Schreiber etal., Int. J. Immunopharmacology 14 (1992), 413-419; Uze et al., J.Interferon Cytokine Res. 15 (1995), 3-26.

The human immunodeficiency virus type 1 (HIV-1) after binding by theenvelope protein gp120 to its cellular receptor CD4, enters susceptiblecells. Several studies have indicated that this gp120-CD4 interactionalone is not sufficient, but that other cellular receptors for viralentry are needed (reviewed in Levy et al., Microbial. Reviews 57 (1993),183-289). Several studies, including inhibition of cell fusion byantibodies (Vanini et al., AIDS 7 (1993), 167-174), peptides (Jiang etal., Nature (London) 365 (1993), 113) and mutation of different sites ingp41 (Cao et al., J. Virol. 67 (1993), 2747-2755) have indicated thatHIV-1 gp41 plays an important role in virus uptake and more general inAIDS pathogenicity. From these studies two regions in addition to theN-terminal aa517-527 have turned out to be of particular importance forcell fusion and viral uptake, namely aa583-599 and aa650-675. Antibodiesagainst regions in SIV gp32 homologous to both regions in HIV-1 gp41could protect macaques from SIV-infection (Shafferman et al., PNAS USA88 (1991), 7126-7130, and Lewis et al., Vaccine 11 (1993), 1347-1355).The first region in gp41 (aa583-599) was demonstrated asimmunosuppressive domain (IS-domain) (Ciancioto et al., Immunol. Lett.19 (1988), 7-14).

The present invention is based on the following observations. Previousstudies demonstrated that HIV-1 gp41 independently of CD4, could bind tohuman T and B cells and monocytes. Thus, it was concluded that ingeneral the target cells (B- and T- cells, monocytes/macrophages)possess, beside CD4, a second receptor on the cell surface which isresponsible for the CD4-independent infection of the cell by theretrovirus. Based on findings of the present inventors that gp41modified MHC antigen expression on these cells and inhibited T cellproliferation, effects similarly shown by human IFN-α and β, but not γ,in the present invention amino acid sequences of gp41 and IFNs werecompared and it was surprisingly found that aa586-596 in gp41 showedsequence similarity with human IFN-α/β receptor binding site in IFN-αand IFN-β. Furthermore, a polyclonal antibody raised to human-IFN-βcould recognise rsgp41 (recombinant soluble glycoprotein 41) from twodifferent sources and its immunosuppressive peptide (ISP, aa583-599) andinhibit, if preincubated with rsgp41, binding of rsgp41 to human cellscompletely. Human IFN-β could partially inhibit the binding of rsgp41 tohuman U937 and Raji cells, but this binding could not be inhibited byIFN-α and IFN-γ. of the gp41 binding proteins isolated by absorptiononto gp41-sepharose four proteins were recognised by binding of IFN-β orby a rabbit anti human IFN-α/β receptor antibody by Western blotanalysis. Absorbing Raji cell solutes on an IFN-β and IFN-α/β-receptorantibody recognised and bound two cellular proteins of 45 and 50 kDawhich were identified as IFN-α/β-receptor proteins; rsg41 and IFN-βcould also recognise two additional proteins of 37 and 62 kDa.Furthermore, anti-human IFN-α/β-receptor-antibodies were able to inhibitHIV infection of PBMC (peripheral blood mononuclear cells). Theseresults indicated that amino acid sequence homology of the receptorbinding regions in gp41 and human IFN-β results in binding to the samecellular proteins and consequently similar functional activity. Thus,the IFN-α/β-receptor may serve as second cellular HIV receptor for gp41binding and inhibiting the interaction between the IFN-α/β-receptor andthe envelope protein of a retrovirus which binds to said receptor, canthus serve as a novel therapeutic approach for preventing or at leastreducing retrovirus infectivity or further propagation of the retrovirusin vivo.

The pharmaceutical compositions comprising the substances of theinvention for inhibiting the above discussed interaction can optionallycomprise a pharmaceutically acceptable carrier or excipient. Examples ofsuitable pharmaceutically acceptable carriers are well known in the artand include phosphate buffered saline solutions, water, emulsions, suchas oil/water emulsions, various types of wetting agents, sterilesolutions etc. Compositions comprising such carriers can be formulatedby well known conventional methods. These pharmaceutical compositionscan be administered to the subject at a suitable dose. Administration ofthe suitable compositions may be effected by different ways, e.g. byintravenous, intraperetoneal, subcutaneous, intramuscular, topical orintradermal administration.

In a preferred embodiment of the pharmaceutical composition of thepresent invention the receptor is the IFN-α/β-receptor.

In another preferred embodiment, the retrovirus is HIV.

In principle, the interaction between the retrovirus and theIFN-receptor can be eliminated by two different approaches, namely byblocking those domains of the IFN-receptor that are responsible for thebinding of retrovirus or, vice versa, by blocking those domains of theretroviral envelope proteins that are responsible for binding to theIFN-receptor.

Accordingly, in a further preferred embodiment the pharmaceuticalcomposition of the present invention comprises a protein, peptide or afunctionally equivalent molecule or a combination thereof which binds tothe IFN-α/β-receptor. Alternatively, the pharmaceutical composition ofthe present invention comprises a protein, peptide or a functionallysimilar molecule or a combination thereof which binds to gp41 of HIV.Such substances can be selected by the person skilled in the art by wellknown methods, for example by blotting, affinity chromatography, otherbinding techniques etc.

In a still further embodiment the pharmaceutical composition comprises aprotein or peptide, which is HIV-gp41, an anti-IFN-α/β-receptor-antibodyor a fragment thereof containing an HIV-gp41 receptor binding site(epitope), or IFN-α or IFN-β or a fragment thereof containing theIFN-α/β-receptor binding site (epitope).

HIV-gp41 can be, for example, recombinantly prepared from clone BH10,described by Vornhagen et al., Biotest Bulletin 4 (1990), 91-96.Anti-IFN-α/β-receptor antibodies can be prepared by well known methodsusing the purified IFN-α/β-receptor as antigen or obtained from SantaCruz Biotechnology, Inc., California, USA. Monoclonal antibodies can beprepared, for example, by the techniques as described in Kohler andMilstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981),3, which comprise the fusion of mouse myeloma cells to spleen cellsderived from immunised mammals. Fragments comprising anHIV-gp41-receptor binding site (epitope) can be also generated byconventional techniques, for example as described by Bogulawski et al.,J. Immunol. Meth. 120 (1983), 51, and Weir (Ed.), Handbook ofExperimental Immunology, Blackwell, Edinburgh, 1986.

IFN-α or IFN-β can be obtained, for example, by Rentschler Biotechnology(Laupheim, Germany) and corresponding fragments still comprising anIFN-α/β-receptor binding site (epitope) can be prepared by well knowntechniques, e.g. enzymatic cleavage, recombinant DNA technology etc.

In an alternative embodiment, the pharmaceutical composition of thepresent invention comprises a soluble IFN-α/β-receptor, ananti-IFN-β-antibody, an anti HIV-gp41-antibody or a fragment of saidproteins or peptides, which contains an HIV-gp41-binding site (epitope).Antibodies and fragments which still bind HIV-gp41 can be obtained bythe methods described above or, for example, from BiosourceInternational (California, USA). A soluble IFN-α/β receptor species canbe created using generally known recombinant DNA-techniques and usingalready published data about the IFN-α/β receptor gene. Omitting thegene(s) segments which enclose the transmembrane and intracellularreceptor domains for instance by using polymerase chain reaction (PCR)technology results in an artificially shortened receptor gene. Thisshortened receptor gene still encodes the ligand binding extracellularand therefore soluble receptor domain. Expressing it in an appropriatesystem leads to a soluble IFN-α/β receptor.

In a particularly preferred embodiment, the pharmaceutical compositioncomprises at least two proteins or peptides binding to theIFN-α/β-receptor or to HIV-gp41 or a combination containing at least oneof the IFN-α/β-receptor binding proteins or peptides and at least one ofthe HIV-gp41 binding proteins. Useful combinations which may haveadditive or synergistic effects and/or lead to a reduction of possibleside effects can be developed by the person skilled in the art byroutine testing. The amount to be administered will depend on the routeof administration, the severity of the infection, the condition of thepatient etc.

In a further preferred embodiment, the pharmaceutical composition of thepresent invention can be used for the prevention or treatment of aretroviral infection, preferably an HIV-infection.

In a still further preferred embodiment, the pharmaceutical compositionadditionally comprises an anti-viral drug. Such a combination mayfurther enhance the effectiveness of said compositions and/or reduceundesired side effects. Examples of suitable anti-viral drugs aredescribed e.g. by Flechter, Am.J.Hosp.Pharm. 51 (1994), 2251-2267, or byStein et al., Clin.Inf.Dis. 17 (1993), 749-771.

Additionally, the pharmaceutical compositions of the present inventionmay contain a ligand for CD4 or a ligand for gp120 surface protein ofHIV. Such a combination may further increase the potency of saidcompositions, since they inhibit the interaction of the retrovirus withsuch target cells exhibiting CD4 on their surface on two differentroutes. Examples of suitable CD4-ligands include gp120 or fragmentsthereof comprising a binding site for CD4.

A further object of the present invention is the use of the aboveproteins, peptides or functionally equivalent molecules for thepreparation of a pharmaceutical composition for the prevention or thetreatment of a retroviral infection, preferably an HIV-infection. Apreferred embodiment relates to the use of said proteins, peptides orfunctionally equivalent molecules in combination with an anti-retroviraldrug and/or a ligand for CD4.

A still further object of the present invention is the use of IFN-β or amolecule having an analogous effect for the preparation of apharmaceutical composition for the prevention or treatment of aretroviral infection by application of a high dose, which is suitablefor the systemic treatment. For such novel use of IFN-β in general dosesin the range of 0.1-1.0×10⁶ International Units (IU) per kg body weightare required. However, the appropriate amounts of IFN-β may varydepending on the particular conditions. Substances that are analogous toIFN-β with respect to the binding to the IFN-α/β-receptor can bedetermined by blotting, affinity chromatography, or other bindingtechniques and include IFN-β fragments, peptides or other designedmolecules recognized by the IFN-α/β receptor.

Finally, the present invention relates to the combination of substancesfor the diagnoses of a HIV infection, based on the detection of theinteraction between HIV-gp41 and the IFN-α/β-receptor.

For instance, a diagnostic procedure to detect HIV infection bydetection of gp41 surface protein could be established as a "sandwichsystem" as follows: A basic layer of IFN-α/β-receptor is able to bindHIV-derived gp41. Bound gp41 is a target for a specific, radioactivelylabelled or dye- or enzyme-coupled antibody (or antibody fragment)permitting a sensitive detection of gp41.

LEGENDS TO THE FIGURES

FIG. 1: Amino acid sequence similarity between HIV-1 gp41 and humanIFN-A and IFN-β receptor binding sites.

A, sequence homology between the first binding site of gp41_(IIIB)(aa583-599, SEQ ID NO: 1) and the IFN-α/β receptor binding region 1 ofIFN-α (SEQ ID NO: 2) and IFN-β (SEQ ID NO: 3) (aa29-35); B, sequencehomology between the first binding site of gp41_(IIIB) (SEQ ID NO: 1)and the IFN-α/β receptor binding region 2 of IFN-α (SEQ ID NO: 4) andIFN-β (SEQ ID NO: 5) (aa123-140).

FIG. 2: Polyclonal sheep antibodies to human IFN-A and IFN-βdose-dependently recognised rsgp41 in an ELISA-assay.

A, polyclonal sheep anti-huIFN-α antibody (immunoglobulin) (4×10³ NU/ml)binding to rsgp41; B, Polyclonal sheep anti-huIFN-β antibody(immunoglobulin) (4×10³ NU/ml) binding to rsgp41; C, two controlantibodies binding to rsgp41 (Ab1: sheep immunoglobulin to human factorI, 1:10, Ab2: normal sheep immunoglobulin, 10 μg/ml).

FIG. 3: Polyclonal sheep anti-human IFN-β antibody recognised gp41IS-peptide (ISP) and two rsgp41 in an ELISA-assay.

A, anti-huIFN-β antibody (immunoglobulin) (4×10³ NU/ml) binding to ISP,P2, CP (control peptide) and their BSA-conjugates as well as EDC-treatedcarrier protein (BSA/EDC); B, anti-huIFN-β antibody (4×10³ NU/ml)binding to two rsgp41 (aa539-684) from Biotest, Germany; aa567-684 fromNEN, USA) (10 μg/mg) and human IFN-β (2×10⁵ U/ml=600 μg/ml).

FIG. 4: a, inhibition of rsgp41 binding to human monocyte cell lineU937, T cell line H9 and B cell line Raji by polyclonal sheep antibodies(immunoglobulin) to human IFN-A and IFN-β in immunofluorescence assay;b. inhibition of rsgp41 binding to U937 by control antibody (normalsheep immunoglobulin)

A, inhibition by sheep anti-huIFN-α antibody; B, inhibition by sheepanti-huIFN-β antibody; C, inhibition by normal sheep immunoglobulin.FACS-histogram overlays: (1) FITC-conjugated Streptavidin (control)(1:50); (2) preincubation of biotinylated rsgp41 (100 ng in 25 μl PBS)with 25 μl PBS for 15 min, then plus FITC-conjugated Streptavidin; (3)preincubation of biotinylated rsgp41 (100 ng) with 25 μl anti-IFN-βantibody (1×10³ NU/ml) or with 25 μl normal sheep immunoglobulin (20μg/ml), then plus FITC-conjugated Streptavidin.

FIG. 5: Inhibition of rsgp41 binding to human cell lines U937, H9 andRaji by human IFN-A, -β and -c in immunofluorescence assay

FACS-histogram overlays: (1) FITC-conjugated Streptavidin (control)(1:50); (2) biotinylated-rsgp41 (100 ng) plus FITC-conjugatedStreptavidin; (3) preincubation of cells with IFN-α, or -β and -c (2×10⁴U/ml), then plus biotinylated rsgp41 (100 ng) and FITC-conjugatedStreptavidin.

FIG. 6: Identification of rsgp41 and IFN-β binding proteins in Raji cellsolutes by Coomassie Blue staining

Raji cell solutes passed through rsgp41, and/or IFN-β sepharose columns(in control, through BSA-column). Eluates were analysed by SDS-PAGE(9.5% gel) under non-reducing conditions. A, molecular weight markers;B, rsgp41-eluate; C, BSA-eluate; D, rsgp41-eluate; E, IFN-β-eluate; F,rsgp41/IFN-β-eluate (rsgp41-eluate passed through IFN-β column). (Eachlane represents an eluate of 1×10⁸ cells).

FIG. 7: Identification of human IFN-β binding proteins in rsgp41 eluateby Western blot

Rsgp41 eluates from Raji cell solutes were subjected to SDS-PAGE (9.5%gel) under non-reducing condition and blotted using IFN-β (1.5×10⁶ U/ml)and three different anti-huIFN-β antibodies (from rabbit, human andmouse) (lane C, E, G) , or using rabbit anti-human IFN-α/β receptorantiserum (lane H, K). Lane B, D, F, I and J are controls (without IFN-βor anti-IFN-α/β-receptor).

FIG. 8: Identification of common cellular proteins in rsgp41 and IFN-βeluates for binding of rsgp41, IFN-β and anti-IFN-β -receptor antibodyby Western blot.

IFN-β eluate and rsgp41/IFN-β eluate from Raji cell solutes weresubjected to SDS-PAGE (9.5% gel) under reducing condition and blottedusing IFN-β (1.5×10⁶ U/ml) plus human anti-huIFN-β antibodies (lane, C,I), or using rabbit anti-human IFN-α/β receptor antiserum (lane E, K),or using rsgp41 plus human anti-gp41 antibody 4BE (lane G, M). Lane B,D, F, H, J and L are controls (without IFN-β or anti-IFN-α/β-receptorand rsgp41). Lanes B-G represented IFN-β eluate and lanes H-Mrepresented rsgp41/IFN-β eluate.

FIG. 9: Rsgp41 from two different sources can bind to human IFN-βbinding proteins in IFN-β-eluate by Western blot.

IFN-β eluate was subjected to SDS-PAGE (9,5% gel) under reducingconditions and blotted using the following probes: lane A, molecularweight markers; lane B, mAb 4B3 control (without rsgp41); lane C, rsgp41(Biotest) plus human anti-gp41 antibody 4B3 (mAb); lane D, rsgp41 (NEN)plus antibody 4B3.

FIG. 10: Inhibition of HIV-infection of human PBMC by rabbitanti-human-IFN-A/β-receptor Polyclonal antibodies (purifiedimmunoglobulins) and antiserum as well as a control antibody (purifiednormal rabbit immunoglobulins).

P24-production tested in p24-ELISA was used as virus replication marker.The concentrations of purified rabbit immunoglobulins are 100 μg/ml. A1:50 dilution corresponds to 2 μg/ml Ig.

The following examples illustrate the invention:

EXAMPLE 1 Anti-IFN-β-antibody recognised rsgp41 and gp41-ISP-region

aa583-599, the immunosuppressive domain of HIV-1 gp41, and aa655-675mediate binding of gp41 to human cells (Chen et al., AIDS 6 (1992),533-539; Qureshi et al., AIDS 4 (1990), 553-558; Henderson and Qureshi,J. Biol. Chem. 268 (1993), 15291-15297). Based on the finding that gp41showed similar effects as human IFN-α and -β, but not -c, on theregulation of MHC molecule expression and the inhibition oflymphoproliferation the amino acid sequence of HIV-1 gp41 was comparedwith human interferons. It was found that aa586-596 showed sequencesimilarity with two regions in human IFN-α and -β (aa29-35 and 123-140),which had been reported to form IFN-α/β receptor binding domains (Fish,J. Interfer. Res. 12 (1992), 257-266, and Fish et al., ibida 9 (1989),97-114). Sequence comparison showed a common three-amino acids epitope(LKD) existing in gp41 (aa594-596) and in the first IFN-α/β receptorbinding region of IFN-α and β (aa29-35) (FIG. 1-A); in addition,aa581-600 of gp41 showed several amino acids identical with those of thesecond IFN-α/β receptor binding region in IFN-β (aa123-140) and in IFN-α(aa117-129) (FIG. 1-B). The second binding site in gp41 (aa655-675) didnot show sequence similarity with human IFN-α and IFN-β.

Based on the fact that there is a common amino acid sequence betweenreceptor binding sites in gp41 and in IFN-α and IFN-β, it was examinedwhether anti-IFN-α or anti-IFN-β antibodies (polyclonal) can recogniseaa539-684 or rsgp41 and gp41 IS-peptide (aa583-599).

Rsgp41 from Biotest (Dreieich, Germany), represents the external portionof the transmembrane protein gp41 of HIV-1 (derived from clone BH10).The soluble transmembrane proteins were expressed in E. coli andpurified. The relative molecular weight of rsgp41 was 18 kD. The bindingof the recombinant proteins to CNBr-Sepharose CL 4B (Pharmacia) wasperformed at a concentration of 1 mg/ml sepharose. Using theBiotin-X-NHS Kit (Calbiochem, No.: 813193; California) and thebiotinylation-method as suggested by Calbiochem, rsgp41 wasbiotin-labeled. HIV-1 gp41 peptide aa567-648 (rsgp41-NEN) was obtainedfrom NEN (DuPont NEN, No.: NEA-211). The gp41 immunosuppressive peptidewas synthesized according to the sequence of the HIV-1 isolate IIIB: HIVaa583-599, LQARILAVERYLKDQQL (SEQ ID NO:1). P2 is a gp41 peptidecontaining the second cellular binding site (Chen et al., AIDS 6 (1992),533-539): HIV-1_(IIIB) Env aa654-677, EESQNQQEKNEQELLELDKWASLW (SEQ IDNO:6). CP is a control peptide with the sequence KDPDAEDASNIMRVISIK (SEQID NO:7), obtained from A. Eberhald (Institute for BiochemicalPharmacology, University Innsbruck, Austria).

Human antiserum (pool) to human IFN-β, rabbit polyclonal and mousemonoclonal anti-human IFN-β antibodies were obtained from RentschlerBiotechnology (Germany). Mouse monoclonal (AB-20-050) and sheeppolyclonal (Ab-19-105) were obtained from Biosource International(California, USA). Human mAb to HIV-1 gp41 (4B3) was obtained from Dr.H. Katinger (Institute of Applied Microbiology, Vienna, Austria).Peroxidase-conjugated rabbit immunoglobulines to mouse immunoglobulines(P161) and to human immunoglobulines (P214), peroxidase-conjugated swineimmunoglobulines to rabbit immunoglobulins (P217) and FITC-conjugatedStreptavidin (F422) were obtained from Dako (Vienna, Austria). Rabbitpolyclonal anti-human IFN-α/β receptor antibody was provided by Dr. D.Novick (Weizmann Institute of Science, Israel).

The sheep anti-human-IFN-β antibody (polyclonal) dose-dependently couldbind to rsgp41 in an ELISA-assay (FIG. 2, B); the sheep anti-human-IFN-αantibody (polyclonal) could bind less well (FIG. 2, A); two controlantibodies (Ab1: sheep immunoglobulin to human factor I: Ab2: normalsheep immunoglobulin) showed background values (FIG. 2, C). The sheepanti-IFN-β antibody could recognise two types of rsgp41 (aa539-684 andaa567-684) from two different sources (Biotest, Germany; Du Pont NEN,USA) (FIG. 3, B), and bind weakly to monomeric gp41 IS-peptide (ISP,aa583-599), but better to ISP conjugated to BSA (ISP-BSA). It couldneither bind to gp41 peptide P2 (aa654-677) and a control peptide (CP)nor to their BSA-conjugates (P2-BSA; CP-BSA) and EDC-treated carrierprotein (BSA/EDC) (FIG. 3, A). (Conjugation was carried out withglobulin-free BSA (Sigma, No. A.7638) using EDC (Sigma, No. E-6383)according to Colgan et al., Current Protocols In Immunology, publishedby John Wiley and Sons, Inc. USA (1991), 9.3.4. Peptide-proteinconjugates were extensively dialysed against phosphate-buffered saline(PBS) before use).

EXAMPLE 2 Anti-IFN-β antibody and human IFN-β block binding of rsgp41 tovarious target cells

U937 is a human monocyte cell line; H9, a CD4⁺ human T cell line; Raji,a human B cell line. These cell lines were grown in RPMI 1640 mediumsupplemented with 10% fetal calf serum (FCS), 2 mmol/l glutamin, 100IU/ml penicillin and 100 μg/ml streptomycin. Since rsgp41 has been shownto bind strongly to human monocyte cell line U937 and B cell line Rajiand weakly to human CD4⁺ T cell line H9 it was examined whether theanti-IFN-α or -β antibodies could block rsgp41 binding to U937, Raji andH9. The results demonstrate that the anti-IFN-β antibody, ifpreincubated with rsgp41, could completely inhibit rsgp41 binding toU937, Raji and H9; the anti-IFN-α antibody showed only a partialblockade (FIG. 4, A); in control, normal sheep immunoglobulin likeanti-IFN-α antibody showed also a partial inhibition of rsgp41 bindingto U937 (FIG. 4, B).

Since the anti-IFN-β antibody could strongly inhibit rsgp41 binding toU937, Raji and H9 cells and IFN-β had considerable sequence homologywith gp41, it was examined whether human IFN-β could inhibit rsgp41binding to these cells by blocking of IFN-α/β-receptors on the cellsurface. The results demonstrated that human IFN-β at a dose of 2×10⁶U/ml, if preincubated with U937, or Raji and H9 cells, could partiallyinhibit rsgp41 binding to the cells, while human IFN-α and c, could notat all influence binding (FIG. 5), showing that inhibition by IFN-β ishighly specific.

EXAMPLE 3 Human IFN-β and HIV-1 share common cellular binding proteins

Since the anti-IFN-β antibody could strongly inhibit rsgp41 binding toU937, Raji and H9 cells and IFN-β had considerable sequence homologywith gp41, it was examined whether human IFN-β could inhibit rsgp41binding to these cells by blocking of IFN-α/β-receptors on the cellsurface. The results demonstrated that human IFN-β at a dose of 2×10⁶U/ml, if preincubated with U937, or Raji and H9 cells, could partiallyinhibit rsgp41 binding to the cells while human IFN-α and IFN-c couldnot at all influence the binding (FIG. 5).

Several cellular binding protein for HIV-1 gp41 were identified in cellsolutes of human T, B and monocyte cells in three laboratories (Chen etal., AIDS 6 (1992), 533-539; Chen et al., Mol. Immunol. 30(1993),1159-1163; Chen et al., Immunol. Letters 37 (1993), 41-45; Ebenbichleret al., AIDS 7 (1993), 489-495; Qureshi et al., AIDS 4 (1990), 553-558;Henderson et al., J. Biol. Chem. 268 (1993), 15291-15297 and Denner etal., J. Can. Res. Clin. Oncol. 121 (S1) (1995), 535 (11/128). To examinewhether IFN-β could recognise similar cellular proteins Raji cellsolutes were passed through a human IFN-β-sepharose column. Theprocedure of protein-coupling to sepharose, cell soluble preparation andadsorption was carried out as described in Chen et al., Mol. Immunol. 30(1993), 1159-1163. Four protein bands of 37, 45, 50 and 62 kDa werealways observed in gp41-eluates by Coomassie blue staining (FIG. 6, laneB and D); sometimes, a few faint bands of 35, 52-60, 80 and 90-100 kDawere seen (FIG. 6, lane D). The eluate from a human IFN-β-sepharosecolumn showed a similar banding pattern with bands at 37, 45, 50 and 62kDa (FIG. 6, lane E). To examine whether IFN-β binding proteins wereidentical with gp41 binding proteins, gp41 eluate from Raji-solute wasadsorbed to and eluated from IFN-β-sepharose column. This eluatecontained the same binding proteins as a gp41 eluate (FIG. 6, lane F).In eluates from Raji-solutes passed through a BSA-sepharose column, noprotein band was seen (FIG. 6, lane C).

EXAMPLE 4 Human IFN-β as well as anti-human IFN-A/β-receptor antibodyrecognised gp41-binding proteins by Western blot

To further elucidate whether gp41 binding proteins and IFN-β bindingproteins were identical it was examined whether anti-human IFN-α/βreceptor antibody and human IFN-β recognised gp41-binding proteins ingp41-eluate from Raji cell solutes by Western blot analysis afterelectrophoresis in 9.5% SDS-PAGE under non-reducing conditions. Beforeprobing, blots were blocked in PBS supplemented with 0.1% gelatine, 1%dried milk, 0.05% Tween-20. Binding of rabbit anti-human IFN-α/βreceptor antibody was detected with peroxidase-conjugatedswine-anti-rabbit IgG (1:100 and 1:500). Normal rabbit serum insteadrabbit anti-IFN-α/β receptor antibody was used as negative control. Fourproteins bands of 45, 50, 57 and 60 kDa were identified in rsgp41-eluateby rabbit anti-human IFN-α/β receptor antiserum (FIG. 7, lane H and K),the 37 kDa band was detected very weakly by using the anti-receptorantiserum, and strongly by a normal rabbit serum (control) (FIG. 7, laneI and J). By ligand blot analysis using IFN-β and three differentantibodies (from human, rabbit and mouse) to human IFN-β, four similaror identical bands of 45, 50, 57 and 60 kDa were blotted (FIG. 7, laneC, E and G). The 57 and 60 kDa band are faint, the binding to bands of37 and 62 kDa was strong, while the rabbit and human (but not mouse mAb,FIG. 7, lane F) antisera to IFN-β alone could weakly bind to these (FIG.7, lane B and D). Besides, a few bands of 66, 80 and 100-120 kDa werealso observed (FIG. 7, lane C, E and G) with IFN-β and anti-IFN-βantibody.

EXAMPLE 5 Rsgp41, human IFN-β and IFN-A/β-receptor antibody recognisedcommon cellular proteins by Western blot

To further examine whether rsgp41 and IFN-β share cellular bindingproteins, we used an IFN-β-eluate (FIG. 8, lanes B-G) from Raji cellsolutes (adsorbed by IFN-β-sepharose column) for Western blot or ligandblot analysis using rsgp41, IFN-β and IFN-α/β-receptor antibody. HumanIFN-β, the anti-human IFN-α/β-receptor antibody and rsgp41 bound to thesame proteins of 45 and 50 kDa in both eluates (FIG. 8, lane C, E, G, I,K and M); in controls (without IFN-β, or rsgp41 and IFN-α/α-R antibody),both proteins were not seen (FIG. 8, lane B, D, F, H, J and L). Thebinding to the bands of 37 and 62 kDa was strong. In controls, the humanantiserum could bind strongly to 62 kDa and weakly to 37 kDa protein(FIG. 8, lane B and H) and normal rabbit serum and human mAb couldweakly bind to 37 kDa protein.

EXAMPLE 6 RSGP41 (from Biotest or NEN) binds to IFN-β-binding-proteins

The binding of rsgp41 from two different sources to IFN-β bindingproteins was examined. The results demonstrated that rsgp41 from Biotestor NEN showed identical binding specifity as IFN-β, namely, rsgp41 couldbind the same proteins of 37, 45, 50 and 62 kDa from IFN-β-eluate (FIG.9, lane C and D), while the human anti-gp41 antibody alone did not (laneB).

EXAMPLE 7 Anti-human-IFN-A/β-receptor-antibodies inhibited HIV-infectionof PBMC

It was examined whether anti-human-IFN-α/β receptor antibodies couldinhibit HIV-infection of human PBMC. PBMC from healthy HIV-seronegativeblood donors were infected with HIV-1_(IIIB). P24-production tested inp24-ELISA was used as virus replication marker (Purtscher et al., AIDSRes. Hum. Retroviruses 10 (1994), 1651-1658). The virus titer wasdetermined by measuring p24 in the supernatant. The assay was performedwith 4 replicates and repeated three times. The results showed that therabbit anti-human-IFN-α/β-receptor polyclonal antibodies (purifiedimmunoglobulins, sc-704, Santa Cruz Biotechnology, Inc.; California,USA) and polyclonal antiserum (obtained from Dr. D. Novick, WeizmannInstitute of Science, Israel) could inhibit HIV-1_(IIIB) -infection ofhuman PBMC; the inhibition by purified antibodies is stronger than bythe antiserum, while purified normal rabbit immunoglobulins as controlantibody did not inhibit HIV-infection (FIG. 10). Normal rabbit serumdid not show any inhibition (data not shown).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - <160> NUMBER OF SEQ ID NOS: 7                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 17                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Source of Artificial Sequ - #ence: synthesized             according to the sequence of the - #the HIV-1 isolate                         IIIB (aa 583-599)                                                        - - <400> SEQUENCE: 1                                                         - - Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Ty - #r Leu Lys Asp Gln Gln        1               5 - #                 10 - #                 15              - - Leu                                                                       - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Source  of Artificial - #Sequence: synthesized             according to the receptor binding - #region 1 of                              human IFN-alpha (aa29-35.)                                               - - <400> SEQUENCE: 2                                                         - - Cys Leu Lys Asp Arg His Asp                                                1               5                                                            - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Source of Artificial Sequ - #ence: synthesized      from                                                                                  receptor binding region 1 of huma - #n IFN-beta (aa                           29-35)                                                                   - - <400> SEQUENCE: 3                                                         - - Cys Leu Lys Asp Arg Met Asn                                                1               5                                                            - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Source of Artificial Sequ - #ence: synthesized     from                                                                                 the human IFN-alpha receptor binding - # region 2 (aa                         123-140)                                                                 - - <400> SEQUENCE: 4                                                         - - Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Ar - #g Ile Thr Leu                1               5 - #                 10                                     - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 18                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Source of Artificial Sequ - #ence: synthesized      from                                                                                  the human IFN-beta receptor binding - #region 2                               (aa123-140)                                                              - - <400> SEQUENCE: 5                                                         - - Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Al - #a Lys Glu Tyr Ser        His                                                                               1               5 - #                 10 - #                 15             - - Cys Ala                                                                   - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 24                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Source of Artificial Sequ - #ence: synthesized      from                                                                                  the HIV-1 IIIB Env protein (aa - #654-677)                               - - <400> SEQUENCE: 6                                                         - - Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Gl - #u Gln Glu Leu Leu        Glu                                                                               1               5 - #                 10 - #                 15             - - Leu Asp Lys Trp Ala Ser Leu Trp                                                       20                                                                - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 18                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Source of Artificial Sequ - #ence: synthesized             control peptide with no function - #or intended sequence                      identity                                                                 - - <400> SEQUENCE: 7                                                         - - Lys Asp Pro Asp Ala Glu Asp Ala Ser Asn Il - #e Met Arg Val Ile Ser        1               5 - #                 10 - #                 15              - - Ile Lys                                                                 __________________________________________________________________________

What is claimed is:
 1. Pharmaceutical composition comprising a protein,peptide or a functionally equivalent molecule or a combination thereofwith the exception of interferon and HIV-gp41 which competitivelyinhibits the binding of a retrovirus to the interferon-(IFN)-receptor ofa target cell and optionally a pharmaceutically acceptable carrier. 2.Pharmaceutical composition according to claim 1 wherein the receptor isthe IFN-α/β-receptor.
 3. Pharmaceutical composition according to claim1, wherein the retrovirus is HIV.
 4. Pharmaceutical compositionaccording to claim 2, wherein the protein, peptide or functionallyequivalent molecule binds to the IFN-α/β-receptor.
 5. Pharmaceuticalcomposition according to claim 4, wherein the protein or peptide is ananti-IFN-α/β-receptor antibody or a fragment of said protein or apeptide containing a HIV-gp41-receptor-binding site (epitope), or afragment of IFN-α or IFN-β containing the IFN-α/β-receptor-binding site(epitope).
 6. Pharmaceutical composition comprising at least 2 proteinsor peptides according to claim
 4. 7. Pharmaceutical compositionaccording to claim 1 for preventing or treating a retroviral infection.8. Pharmaceutical composition according to claim 7, wherein theretroviral infection is an HIV infection.
 9. Pharmaceutical compositionaccording to claim 1, additionally comprising an anti-retroviral drug.10. Pharmaceutical composition according to claim 1, further comprisinga ligand to CD4, or a ligand to gp120 surface protein of HIV.
 11. Amethod for treatment of a retroviral infection comprising administratingto a patient in need thereof a protein, peptide or a functionallyequivalent molecule which competitively inhibits the binding of aretrovirus to the IFN-receptor of a target cell by blocking thosedomains of the IFN-receptor that are responsible for the binding of aretrovirus or by blocking those domains of the retroviral envelopeproteins that are responsible for binding to the IFN-receptor.
 12. Themethod according to claim 11, wherein the receptor is theIFN-α/β-receptor.
 13. The method according to claim 11, wherein theretrovirus is HIV.
 14. The method according to claim 12, wherein theprotein, peptide or functionally equivalent molecule binds to HIV-gp41.15. The method according to claim 12, wherein the protein, peptide orfunctionally equivalent molecule binds to the IFN-α/β-receptor.
 16. Themethod according to claim 15, wherein the protein or peptide isHIV-gp41, and anti-IFN-α/β-receptor antibody or a fragment of saidprotein or peptide containing a HIV-gp41-receptor-binding site(epitope), or IFN-α- or IFN-β- or a fragment thereof containing theIFN-α/β-receptor-binding site (epitope).
 17. The method according toclaim 14, wherein the protein or peptide is a soluble IFN-α/β-receptor,an anti-IFN-β-antibody, an anti-HIV-gp41-antibody or a fragment of saidprotein or peptide containing an HIV-gp-41-binding site (epitope). 18.The method according to claim 11, wherein the pharmaceutical compositioncomprises a ligand to CD4, or a ligand to gp120 surface protein of HIV.19. The method according to claim 11, wherein the protein, peptide orfunctionally equivalent molecule is additionally combined with ananti-retroviral drug and/or ligand to CD4.
 20. A method for treatment ofa retroviral infection comprising administrating to a patient in needthereof a protein, peptide or a functionally equivalent molecule whichbinds to HIV-gp41 and which competitively inhibits the binding of aretrovirus to the IFN-α/β-receptor of a target cell by blocking thosedomains of the IFN-α/β-receptor that are responsible for the binding ofa retrovirus or by blocking those domains of the retroviral envelopeproteins that are responsible for binding to the IFN-α/β-receptor.
 21. Amethod for treatment of a retroviral infection comprising administratingto a patient in need thereof a protein, peptide or a functionallyequivalent molecule which binds to HIV-gp41 and which competitivelyinhibits the binding of a retrovirus to the IFN-α/β-receptor of a targetcell by blocking those domains of the IFN-α/β-receptor that areresponsible for the binding of a retrovirus or by blocking those domainsof the retroviral envelope proteins that are responsible for binding tothe IFN-α/β-receptor, wherein said protein, peptide or functionallyequivalent molecule is a soluble molecule selected from the groupconsisting of the IFN-α/β-receptor, an anti-IFN-α/β-receptor, ananti-HIV-gp41-antibody and a fragment of said protein that contains anHIV-gp41-binding site (epitope).
 22. A method of treatment comprisingadministering to a patient in need thereof an effective amount of apharmaceutical composition comprising a protein, peptide or afunctionally equivalent molecule or a combination thereof with theexception of interferon and HIV-gp41, which competitively inhibits thebinding of a retrovirus to the interferon-(IFN)-receptor of a targetcell and optionally a pharmaceutically acceptable carrier.
 23. Themethod according to claim 22, wherein the receptor is theIFN-α/β-receptor.
 24. The method according to claim 22, wherein theretrovirus is HIV.
 25. The method according to claim 23, wherein theprotein, peptide or functionally equivalent molecule binds to theIFN-α/β-receptor.
 26. The method according to claim 25, wherein theprotein or peptide is an anti-IFN-α/β-receptor antibody or a fragment ofsaid protein or a peptide containing a HIV-gp41-receptor-binding site(epitope), or a fragment of IFN-α or IFN-β containing theIFN-α/β-receptor-binding site (epitope).
 27. The method according toclaim 25, wherein said pharmaceutical composition comprises at least twoproteins or peptides and wherein said proteins bind to theIFN-α/β-receptor.
 28. The method according to claim 22, wherein thepharmaceutical composition additionally comprises and anti-retroviraldrug.
 29. The method according to claim 22, wherein the pharmaceuticalcomposition further comprises a ligand to CD4, or a ligand to gp120surface protein of HIV.
 30. A method for inhibiting or treating aretroviral infection comprising administrating to a patient in needthereof a protein, peptide or a functionally equivalent molecule orcombination thereof, with the exception of IFN-α, which binds toHIV-gp41 and which competitively inhibits the binding of a retrovirus tothe IFN-α/β-receptor of a target cell by blocking those domains of theIFN-α/β-receptor that are responsible for the binding of a retrovirus orby blocking those domains of the retroviral envelope proteins that areresponsible for binding to the IFN-α/β-receptor.